Circuits for the control of output current in an electronic device for performing active biological operations

ABSTRACT

A circuit for control of an output current in a multiple unit cell array includes an array of unit cells arranged in rows and columns. Each unit cell includes a column select transistor being adapted for control by a column selector and a row select transistor being adapted for control by a row selector. The column select transistor and the row select transistor are connected together in series to each other and between an output node and a first supply. A return electrode is provided to complete the circuit.

RELATED APPLICATION INFORMATION

[0001] This application is a continuation application of U.S.application Ser. No. 09/239,598, filed Jan. 29, 1999, which is acontinuation-in-part application of application Ser. No. 09/026,618,filed Feb. 20, 1998, entitled “Advanced Active Electronic Devices forMolecular Biological Analysis and Diagnostics and Methods forManufacture of Same”, now issued as U.S. Pat. No. 6,099,803, which is acontinuation-in-part of application Ser. No. 08/753,962, filed Dec. 4,1996, entitled “Laminated Assembly for Active Bioelectronic Devices”,which is a continuation-in-part of Ser. No. 08/534,454, filed Sep. 27,1995, entitled “Apparatus and Methods for Active Programmable MatrixDevices”, now issued as U.S. Pat. No. 5,849,486, which is acontinuation-in-part of application Ser. No. 08/304,657, filed Sep. 9,1994, entitled, as amended, “Molecular Biological Diagnostic SystemsIncluding Electrodes”, now issued as U.S. Pat. No. 5,632,957, continuedas Ser. No. 08/859,644, filed May 20, 1997, entitled “Control System forActive Programmable Electronic Microbiology System” which is acontinuation-in-part of application Ser. No. 08/271,882, filed Jul. 7,1994, entitled, as amended, “Methods for Electronic Stringency Controlfor Molecular Biological Analysis and Diagnostics”, now issued as U.S.Pat. No. 6,017,696, which is a continuation-in-part of application Ser.No. 08/146,504, filed Nov. 1, 1993, entitled, as amended, “ActiveProgrammable Electronic Devices for Molecular Biological Analysis andDiagnostics”, now issued as U.S. Pat. No. 5,605,662, continued asapplication Ser. No. 08/725,976, filed Oct. 4, 1996, entitled “Methodsfor Electronic Synthesis of Polymers”, now issued as U.S. Pat. No.5,929,208, and application Ser. No. 08/709,358, filed Sep. 6, 1996,entitled “Apparatus and Methods for Active Biological SamplePreparation”, now issued as U.S. Pat. No. 6,129,828, and is related toapplication Ser. No. 08/677,305, filed Jul. 9, 1996, entitled“Multiplexed Active Biological Array”, now issued as U.S. Pat. No.5,965,452, and is also related to application Ser. No. 08/846,876, filedMay 1, 1997, entitled “Scanning Optical Detection System”, allincorporated herein by reference as if fully set forth herein.

[0002] This application is also related to the following applicationsfiled on Jan. 29, 1999: application Ser. No. 09/240,489, entitled“Advanced Active Electronic Devices Including Collection Electrodes forMolecular Biological Analysis and Diagnostics”, now issued as U.S. Pat.No. 6,225,059, U.S. application Ser. No. 09/239,569 entitled“Multicomponent Devices for Molecular Biological Analysis andDiagnostics”, now issued as U.S. Pat. No. 6,068,818, U.S. applicationSer. No. 09/240,920 entitled “Methods for Fabricating MulticomponentDevices for Molecular Biological Analysis and Diagnostics”, now allowed,U.S. application Ser. No. 09/240,931 entitled and “Devices for MolecularBiological Analysis and Diagnostics Including Waveguides”, all of whichare incorporated herein by reference.

FIELD OF THE INVENTION

[0003] The invention relates to circuits useful in performing activebiological operations. More particularly, the invention relates tocircuits for the control of output current in an electronic device forperforming active biological operations.

BACKGROUND OF THE INVENTION

[0004] Molecular biology comprises a wide variety of techniques for theanalysis of nucleic acid and protein. Many of these techniques andprocedures form the basis of clinical diagnostic assays and tests. Thesetechniques include nucleic acid hybridization analysis, restrictionenzyme analysis, genetic sequence analysis, and the separation andpurification of nucleic acids and proteins (See, e.g., J. Sambrook, E.F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2Ed., Cold spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989).

[0005] Most of these techniques involve carrying out numerous operations(e.g., pipetting, centrifugations, electrophoresis) on a large number ofsamples. They are often complex and time consuming, and generallyrequire a high degree of accuracy. Many a technique is limited in itsapplication by a lack of sensitivity, specificity, or reproducibility.For example, these problems have limited many diagnostic applications ofnucleic acid hybridization analysis.

[0006] The complete process for carrying out a DNA hybridizationanalysis for a genetic or infectious disease is very involved. Broadlyspeaking, the complete process may be divided into a number of steps andsubsteps. In the case of genetic disease diagnosis, the first stepinvolves obtaining the sample (blood or tissue). Depending on the typeof sample, various pre-treatments would be carried out. The second stepinvolves disrupting or lysing the cells, which then release the crudeDNA material along with other cellular constituents. Generally, severalsub-steps are necessary to remove cell debris and to purify further thecrude DNA. At this point several options exist for further processingand analysis. One option involves denaturing the purified sample DNA andcarrying out a direct hybridization analysis in one of many formats (dotblot, microbead, microplate, etc.). A second option, called Southernblot hybridization, involves cleaving the DNA with restriction enzymes,separating the DNA fragments on an electrophoretic gel, blotting to amembrane filter, and then hybridizing the blot with specific DNA probesequences. This procedure effectively reduces the complexity of thegenomic DNA sample, and thereby helps to improve the hybridizationspecificity and sensitivity. Unfortunately, this procedure is long andarduous. A third option is to carry out the polymerase chain reaction(PCR) or other amplification procedure. The PCR procedure amplifies(increases) the number of target DNA sequences relative to non-targetsequences. Amplification of target DNA helps to overcome problemsrelated to complexity and sensitivity in genomic DNA analysis. All theseprocedures are time consuming, relatively complicated, and addsignificantly to the cost of a diagnostic test. After these samplepreparation and DNA processing steps, the actual hybridization reactionis performed. Finally, detection and data analysis convert thehybridization event into an analytical result.

[0007] The steps of sample preparation and processing have typicallybeen performed separate and apart from the other main steps ofhybridization and detection and analysis. Indeed, the various substepscomprising sample preparation and DNA processing have often beenperformed as a discrete operation separate and apart from the othersubsteps. Considering these substeps in more detail, samples have beenobtained through any number of means, such as obtaining of full blood,tissue, or other biological fluid samples. In the case of blood, thesample is processed to remove red blood cells and retain the desirednucleated (white) cells. This process is usually carried out by densitygradient centrifugation. Cell disruption or lysis is then carried out onthe nucleated cells to release DNA, preferably by the technique ofsonication, freeze/thawing, or by addition of lysing reagents. Crude DNAis then separated from the cellular debris by a centrifugation step.Prior to hybridization, double-stranded DNA is denatured intosingle-stranded form. Denaturation of the double-stranded DNA hasgenerally been performed by the techniques involving heating (>Tm),changing salt concentration, addition of base (NaOH), or denaturingreagents (urea, formamide, etc.).

[0008] Nucleic acid hybridization analysis generally involves thedetection of a very small number of specific target nucleic acids (DNAor RNA) with an excess of probe DNA, among a relatively large amount ofcomplex non-target nucleic acids. The substeps of DNA complexityreduction in sample preparation have been utilized to help detect lowcopy numbers (i.e. 10,000 to 100,000) of nucleic acid targets. DNAcomplexity is overcome to some degree by amplification of target nucleicacid sequences using polymerase chain reaction (PCR). (See, M. A. Inniset al, PCR Protocols: A Guide to Methods and Applications, AcademicPress, 1990). While amplification results in an enormous number oftarget nucleic acid sequences that improves the subsequent direct probehybridization step, amplification involves lengthy and cumbersomeprocedures that typically must be performed on a stand alone basisrelative to the other substeps. Substantially complicated and relativelylarge equipment is required to perform the amplification step.

[0009] The actual hybridization reaction represents one of the mostimportant and central steps in the whole process. The hybridization stepinvolves placing the prepared DNA sample in contact with a specificreporter probe, at a set of optimal conditions for hybridization tooccur to the target DNA sequence. Hybridization may be performed in anyone of a number of formats. For example, multiple sample nucleic acidhybridization analysis has been conducted on a variety of filter andsolid support formats (See G. A. Beltz et al., in Methods in Enzymology,Vol. 100, Part B, R. Wu, L. Grossman, K. Moldave, Eds., Academic Press,New York, Chapter 19, pp. 266-308, 1985). One format, the so-called “dotblot” hybridization, involves the non-covalent attachment of target DNAsto filter, which are subsequently hybridized with a radioisotope labeledprobe(s). “Dot blot” hybridization gained wide-spread use, and manyversions were developed (see M. L. M. Anderson and B. D. Young, inNucleic Acid Hybridization—A Practical Approach, B. D. Hames and S. J.Higgins, Eds., IRL Press, Washington, D.C. Chapter 4, pp. 73-111, 1985).It has been developed for multiple analysis of genomic mutations (D.Nanibhushan and D. Rabin, in EPA 0228075, Jul. 8, 1987) and for thedetection of overlapping clones and the construction of genomic maps (G.A. Evans, in U.S. Pat. No. 5,219,726, Jun. 15, 1993).

[0010] New techniques are being developed for carrying out multiplesample nucleic acid hybridization analysis on micro-formatted multiplexor matrix devices (e.g., DNA chips) (see M. Barinaga, 253 Science, pp.1489, 1991; W. Bains, 10 Bio/Technology, pp.757-758, 1992). Thesemethods usually attach specific DNA sequences to very small specificareas of a solid support, such as micro-wells of a DNA chip. Thesehybridization formats are micro-scale versions of the conventional “dotblot” and “sandwich” hybridization systems.

[0011] The micro-formatted hybridization can be used to carry out“sequencing by hybridization” (SBH) (see M. Barinaga, 253 Science, pp.1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). SBH makesuse of all possible n-nucleotide oligomers (n-mers) to identify n-mersin an unknown DNA sample, which are subsequently aligned by algorithmanalysis to produce the DNA sequence (R. Drmanac and R. Crkvenjakov,Yugoslav Patent Application #570/87, 1987; R. Drmanac et al., 4Genomics, 114, 1989; Strezoska et al., 88 Proc. Natl. Acad. Sci. USA10089, 1992; and R. Drmanac and R. B. Crkvenjakov, U.S. Pat. No.5,202,231, Apr. 13, 1993).

[0012] There are two formats for carrying out SBH. The first formatinvolves creating an array of all possible n-mers on a support, which isthen hybridized with the target sequence. The second format involvesattaching the target sequence to a support, which is sequentially probedwith all possible n-mers. Both formats have the fundamental problems ofdirect probe hybridizations and additional difficulties related tomultiplex hybridizations.

[0013] Southern, United Kingdom Patent Application GB 8810400, 1988; E.M. Southern et al., 13 Genomics 1008, 1992, proposed using the firstformat to analyze or sequence DNA. Southern identified a known singlepoint mutation using PCR amplified genomic DNA. Southern also describeda method for synthesizing an array of oligonucleotides on a solidsupport for SBH. However, Southern did not address how to achieveoptimal stringency condition for each oligonucleotide on an array.

[0014] Concurrently, Drmanac et al., 260 Science 1649-1652, 1993, usedthe second format to sequence several short (116 bp) DNA sequences.Target DNAs were attached to membrane supports (“dot blot” format). Eachfilter was sequentially hybridized with 272 labeled 10-mer and 1-meroligonucleotides. A wide range of stringency condition was used toachieve specific hybridization for each n-mer probe; washing timesvaried from 5 minutes to overnight, and temperatures from 0° C. to 16°C. Most probes required 3 hours of washing at 16° C. The filters had tobe exposed for 2 to 18 hours in order to detect hybridization signals.The overall false positive hybridization rate was 5% in spite of thesimple target sequences, the reduced set of oligomer probes, and the useof the most stringent conditions available.

[0015] A variety of methods exist for detection and analysis of thehybridization events. Depending on the reporter group (fluorophore,enzyme, radioisotope, etc.) used to label the DNA probe, detection andanalysis are carried out fluorimetrically, calorimetrically, or byautoradiography. By observing and measuring emitted radiation, such asfluorescent radiation or particle emission, information may be obtainedabout the hybridization events. Even when detection methods have veryhigh intrinsic sensitivity, detection of hybridization events isdifficult because of the background presence of non-specifically boundmaterials. A number of other factors also reduce the sensitivity andselectivity of DNA hybridization assays.

[0016] Attempts have been made to combine certain processing steps orsubsteps together. For example, various microrobotic systems have beenproposed for preparing arrays of DNA probe on a support material. Forexample, Beattie et al., in The 1992 San Diego Conference: GeneticRecognition, November, 1992, used a microrobotic system to depositmicro-droplets containing specific DNA sequences into individualmicrofabricated sample wells on a glass substrate.

[0017] Generally, the prior art processes have been extremely labor andtime intensive. For example, the PCR amplification process is timeconsuming and adds cost to the diagnostic assay. Multiple stepsrequiring human intervention either during the process or betweenprocesses is suboptimal in that there is a possibility of contaminationand operator error. Further, the use of multiple machines or complicatedrobotic systems for performing the individual processes is oftenprohibitive except for the largest laboratories, both in terms of theexpense and physical space requirements.

[0018] Attempts have been made to enhance the overall sampleintroduction, to sample preparation analysis process. Given therelatively small volume of sample material which is often timesavailable, improved processes are desired for the efficient provisionsof sample, transport of sample and effective analysis of sample. Whilevarious proposals have been advanced, certain systems enjoy relativeadvantages in certain circumstances.

[0019] Yet another area of interest is in the electrical addressing ofrelatively large arrays. As array grow relatively large, the efficientoperation of the system becomes more of a consideration. Efficientinterfacing of an array based system with electrical connectionsoff-chip raise pin or contact limitation issues. Further, constraintsregarding effective chip or array size present issues regarding theselection of components, and the size of them, for inclusion on the chipor substrate. Often times, various selections must be made to provide aneffective optimization of advantages in the overall design.

[0020] One proposed solution for the control of an array of electrodesutilizing less than one individual dedicated connection per electrode ortest site is provided in Kovacs U.S. patent application Ser. No.08/677,305, entitled “Multiplexed Active Biological Array”, filed Jul.9, 1996, incorporated herein as if fully set forth herein. The array isformed of a plurality of electrode sites, a typical electrode siteincluding an electrode, a driving element coupled to the electrode forapplying an electrical stimulus to the electrode and a local memorycoupled to the driving element for receiving and storing a signalindicative of a magnitude of the electrical stimulus to be applied tothe electrode. Multiple embodiments are disclosed for selectivelycoupling a value signal through coaction of a row line and a column linefor storage in the local memory. In this way, the values at the variouselectrodes in the array may differ from one another.

[0021] In Fiaccabrino, G. C., et al., “Array of Individual AddressableMicroelectrodes”, Sensors and Actuators B, 18-19, (1994) 675-677, anarray of n² electrodes are connected to two n pins, plus 2 additionalpins for signal output and bulk bias. The row and column signals driveseries connected transistors to provide a single value to a workingelectrode. This system does not enable the switching of two or moreelectrodes simultaneously at different potentials.

[0022] In Kakerow, R. et al., “A Monolithic Sensor Array of IndividuallyAddressable Microelectrodes”, Sensors and Actuators A, 43 (1994)296-301, a monolithic single chip sensor array for measuring chemicaland biochemical parameters is described. A 20×20 array of individuallyaddessable sensor cells is provided. The sensor cells are seriallyaddressed by the sensor control unit. One horizontal and one verticalshift register control selection of the sensor cells. Only one sensorcell is selected at a time. As a result, multiple sites may not beactivated simultaneously.

[0023] Yet another concern is the ability to test an electronic deviceprior to application of a conductive solution on the device. As devicesor chips become more complicated, the possibility of a manufacturing orprocess error generally increases. While visual inspection of circuitrymay be performed, further testing may ensure an operational device isprovided to the end user.

[0024] As is apparent from the preceding discussion, numerous attemptshave been made to provide effective techniques to conduct multi-step,multiplex molecular biological reactions. However, for the reasonsstated above, these techniques are “piece-meal”, limited and have noteffectively optimized solutions. These various approaches are not easilycombined to form a system which can carry out a complete DNA diagnosticassay. Despite the long-recognized need for such a system, nosatisfactory solution has been proposed previously.

SUMMARY OF THE INVENTION

[0025] Methods of manufacture and apparatus adapted for advantageous usein active electronic devices utilized for biological diagnostics aredisclosed. Specifically, various layouts or embodiments, including theselection of components, are utilized in advantageous combination toprovide useful devices. Various structures, shapes and combinations ofelectrodes coact with various applied signals (voltages, currents) so asto effect useful preparation, transport, diagnosis, and analysis ofbiological or other electrically charged material. Various advantageousprotocols are described.

[0026] In a first preferred embodiment, an electronic device forperforming active biological operations comprises in combination asupport substrate, an array of microlocations disposed on the substrate,a first collection electrode disposed on the substrate, first and secondfocusing electrodes disposed on the substrate, the first and secondelectrodes being disposed at least in part adjacent the array ofmicrolocations, the distance between the first and second electrodesadjacent the array preferably being smaller than the distance betweenthe first and second electrodes in yet another region disposed away fromthe array, and counter electrodes disposed on the substrate. In oneimplementation, a “V” or “Y” configuration is utilized, which serves tofocus charged biological material into a desired region. Preferably, thefocusing electrodes have a proximal end disposed near or adjacent thearray of microlocations, and a remote portion disposed away from thearray. The distance between the proximal ends of the first and secondelectrode is less than the distance between the proximal ends of thefirst and second electrode.

[0027] In operation of this embodiment, a solution containing DNA orother biological material to be interrogated is provided to the device,above the substrate. As a typical initial step, the concentrationelectrode and return electrodes are activated so as to transport andconcentrate the charged biological materials onto or near theconcentration region. In the preferred embodiment, the concentrationelectrode and the return electrode or electrodes interrogate arelatively large volume of the sample. Typically, the collectionelectrode and counter electrodes are disposed on the substrate so thatthe electrophoretic lines of force are significant over substantiallyall of the flow cell volume. By way of example, the concentration andreturn electrodes may be disposed near the periphery of the footprint ofthe flowcell. In yet another embodiment, they are maybe disposed atsubstantially opposite ends of the flow cell. In yet another embodiment,the return electrode substantially circumscribes the footprint of theflow, with a centrally disposed collection electrode. Effectiveinterrogation of the sample within the flow cell is one desired result.Once the sample has been corrected, the focusing electrodes may beoperated so as to funnel or further focus the materials towards thearray of microlocations. As materials move from the concentrationelectrode towards the array, the decreasing spacing between the firstand second focusing electrodes serves to concentrate the analytes andother charged material into a smaller volume. In this way, a moreeffective transportation of materials from a relatively largerconcentration electrode region to a relatively smaller microelectrodearray region may be achieved.

[0028] It yet another optional aspect of this embodiment of thisinvention, one or more transport electrodes are provided, the transportelectrodes being disposed on the substrate, and positioned between thefirst collection electrode and the array. In the preferred embodiment,there are at least two transport electrodes, and further, the transportelectrodes are of a different size, preferably wherein the ratio oflarger to smaller is at least 2:1. In this way, the relatively largearea subtended by the collection electrode may be progressively moved tosmaller and smaller locations near the analytical region of the device.This arrangement both aids in transitioning from the relatively largearea of the collection electrode, but the stepped nature of theembodiment reduces current density mismatches. By utilizing a stepped,preferably monotonically stepped size reduction, more effectivetransportation and reduced burnout are achieved.

[0029] In yet another embodiment of device, an electronic device forperforming biological operations comprises a support substrate, an arrayof microlocations disposed on the substrate, the array being formedwithin a region, the region including a first side and an opposite side,a first collection electrode disposed on the substrate adjacent thearray, and a second collection electrode disposed on the substrate,adjacent the array, the first and second collection electrodes being atleast in part on the opposite side of the region. In the preferredembodiment, the collection electrodes have an area at least 80% of thearea of the region of the array. In this way, the sample may becollected in a relatively large area adjacent the region containingmicrolocations, from which the DNA or other charged biological materialsmay be provided to the region.

[0030] In one method for use of this device, the collection electrodemay first collect the materials, and then be placed repulsive relativeto the collected material, thereby sweeping the material towards theregion containing the array. The material may be transported in a wavemanner over the array, permitting either interaction with a passivearray or an electrically active array. Alternatively, the material maybe moved over the region of the array, and effective maintained in thatposition by application of AC fields. This embodiment has proved capableof performance of repeat hybridizations, where material is move to andinteracted with the array, after which it is moved out of the region,and preferably held by the collection electrode or on another electrode,after which it is moved to the array for a second, though possiblydifferent, interaction.

[0031] In yet another embodiment of device design, a substantiallyconcentric ring design is utilized. In combination, an electronic devicefor performing active biological operations includes a supportsubstrate, an array of microlocations disposed on the substrate in aannular region, a first counter electrode disposed on the substratesurrounding the array, and a collection electrode disposed on thesubstrate and disposed interior of the array. In the preferredembodiment, the first counter or return electrode is segmented,optionally having pathways resulting in the segmentation which serve aspathways for electrical connection to the array. In yet anothervariation of this embodiment, multiple rings are provided surroundingthe array.

[0032] In yet another embodiment of this invention, a reduced componentcount, preferably five component, system is implemented in a flip-chiparrangement for providing active biological diagnostics. The devicecomprises in combination a support substrate having first and secondsurfaces and a via, pathway or hole between the first and secondsurfaces to permit fluid flow through the substrate, at least one of thefirst and second surfaces supporting electrical traces, a secondsubstrate including at least a first surface, the first surface beingadapted to be disposed in facing arrangement with at least one of thefirst and second surfaces of the first substrate and positioned near,e.g., under, the via, the second substrate including electricallyconductive traces connecting to an array of microlocations, the arraybeing adapted to receive said fluid through the via, pathway or hole,electrically conductive interconnects, e.g., bumps, interconnecting theelectrical traces on the second surface of the support substrate and theelectrical traces on the first surface of the second substrate, asealant disposed between the second face of the support substrate andthe first face of the second substrate, said sealant providing a fluidicseal by and between the first substrate and the second substrate, andoptionally, a flowcell dispose on the first surface of the firstsubstrate. Preferably, the structures utilize a flip-chip arrangement,with the diagnostic chip below the support substrate in operationalorientation. This design is particularly advantageous in reducing thenumber of components in the device, and to improve manufacturingreliability.

[0033] In yet another embodiment, an electronic device for performingactive biological operations comprises a support substrate having afirst and second surface, and a via between the first and secondsurfaces to permit fluid flow through the substrate, a second substrateincluding at least a first surface, the first surface being adapted tobe disposed in facing arrangement with the second surface of the firstsubstrate, the second substrate including an array of microlocations,the array being adapted to receive said fluid, a sealant disposedbetween the second face of the support substrate and the first face ofthe second substrate, a source of illumination, and a waveguide havingan input adapted to receive the illumination from the source, and anoutput adapted to direct the illumination to the array, the waveguidebeing substantially parallel to the support substrate, the illuminationfrom the waveguide illuminating the array. In the preferred embodiment,the source of illumination is a laser, such as a laser bar. Such adevice may utilize a support substrate which is flex circuit or acircuit board.

[0034] A novel, advantageous method of manufacture may be utilized withsome or all of the embodiments. The method is particularly advantageousfor the manufacture of the flip-chip design. In that structure, there isa chip disposed adjacent a substrate, the substrate including a viatherethrough, the structure being adapted to receive a fluid to beplaced on the substrate, and to flow through the via down to the chip,where at least a portion of the chip includes an area to be free ofsealant overcoat. Selection of sealant viscosity and materials mayeffectively result in effective coverage, good thermal contact betweenthe substrate and the chip, and fluidic sealing. In the most preferredembodiment, the method may include use of a light-curable sealant whichis cured with light during application. Specifically, light is exposedto the device onto the substrate and through the via, down to the chip.Next, a light curable, wickable sealant is applied to the interfacebetween the substrate and the chip. The light at least partially curesthe sealant as a result of the exposure, whereby the sealant isprecluded from flowing to said area to be free of sealant. Finally, ifdesired, the cure of the sealant may be completed, such as by heattreatment.

[0035] In yet another embodiment, a system or chip includes a multi-sitearray with electrically repetitive unit cell locations. Typically, thearray is formed of rows and columns, most typically an equal number ofrows and columns. The individual unit cells of the array of unit cellsis selected by action of selectors such as one or more row selectors andone or more column selectors. The selector may be a memory, such as ashift register memory, or a decoder, or a combination of both. An inputfor address information receives addresses, typically from off-chip,though on-chip address generators may be utilized. In the preferredembodiment, the row selectors comprise shift registers, either in a byone configuration, or in a wider configuration, such as a by fourconfiguration. In operation, the selection registers are sequentiallyloaded with values indicating the selection or non-selection of a unitcell, and optionally, the value (or indicator of value) of output forthat cell. Optionally, memory may be provided to retain those values soas to continue the output from the unit cell.

[0036] The system or chip provides for the selective provision ofcurrent and voltage in an active biological matrix device which isadapted to receive a conductive solution including charged biologicalmaterials. In one aspect, an array of unit cells is provided. Each unitcell typically includes a row contact and a column contact. Row linesare disposed within the array, the row lines being coupled to the rowcontacts of the unit cell. A row selector selectively provides a rowselect voltage to the row lines. Further, column lines are disposedwithin the array, the column lines being coupled to the column contactsof the array. A column selector selectively provides a column selectsignal to the column lines. The unit cells are coupled to a supplyvoltage and to an electrode, the row select signal and the column selectsignal serving to select a variable current output from the electrode ofthe unit cell. A return electrode is coupled to a potential and adaptedto contact the conductive solution. In operation, selective activationof one or more unit cells results in the provision of current within theconductive solution.

[0037] In one preferred embodiment of a unit cell, a symmetricarrangement is utilized. A first column select unit, preferably atransistor, and a first row select unit, also preferably a transistor,are in series relation between a first source, e.g., voltage and/orcurrent source, and a node, typically a current output node. In thepreferred embodiment, the column select transistor may be preciselycontrolled under application of a gate voltage such as from the columnshift register memory. Preferably, the select units may differ from eachother in their controllability, such as by varying the channel length inthe control transistor. The channel lengths have been chosen so as tomatch the gain or other desired properties between the row and columntransistors. Also, the long channel length provides the ability tocontrol small currents with reasonable control signals. Thus, byapplication of potentials from the row selector and column selector,application of potential to the control gates results in output ofcurrent at the unit cell.

[0038] The unit cell circuit preferably further includes a second columnselect unit, preferably a transistor, and a second row select unit, alsopreferably a transistor, used in series relation between a secondsource, e.g., voltage and/or current source, and a node, typically thepreviously referred to node, i.e., a current output node. In thepreferred embodiment, the first source is a supply potential Vcc and thesecond source is a reference potential, such as ground. Preferably thenodes are the same node, such that there is a series connection betweenVcc and ground of the first column select unit and first row selectunit, the node, and the second row select unit and the second columnselect unit. Optionally, the return electrode is biased at a potentialbetween the potential of the first source and the second source, e.g.,Vcc/2.

[0039] In yet another aspect of the preferred embodiment, test circuitryis included. Test circuitry may be utilized to ensure circuitcontinuity, by permitting testing prior to application of a fluidicsolution. A first test transistor spans the first column select andfirst row select transistor. Likewise, a second test transistor spansthe second column select and second row select transistor. Selectiveactivation ensures continuity of the circuit. Alternatively, the testcircuit function may be performed by special programming of the row andcolumn transistors, e.g., turning on of the first and second row selectand first and second column select transistors.

[0040] In yet a further aspect of this invention, the current supply tothe test site is varied. Examples of the variation of current over timemay include static direct current (i.e., no variation as a function oftime), square wave, sinusoidal, sawtooth, or any waveform which varieswith time. In one embodiment, the currents, whether static or varying asa function of time, are supplied to the column selection circuitry,which are then selectively provided in a digital manner to the columnlines for coupling to the selected electrodes. This mixed analog anddigital technique permits significant control of the values andwaveforms of the current supplied at the individual electrodes. Thewaveforms, e.g., the current waveforms, may be generated either on-chipor off-chip. Additionally, control or operation of the overallcircuitry, and/or generation of signals such as the current waveformsmay be generated through the use of digital to analog converters (DACs),central processing units (CPUs), through the use of local memory forstorage of values, through the use of clock generators for timing andcontrol of various waveforms, and through the use of digital signalprocessors (DSPs).

[0041] In one aspect of this invention, a system based upon currentcontrol of a first current is utilized to effect control of a secondcurrent. Preferably, a current mirror arrangement is utilized. A currentsupply provides a variable value of current for use in a voltagegeneration circuit. In the preferred embodiment, multiple currentsources are utilized, being summed at their output, under the selectivecontrol of a memory for selective inclusion. A variable voltage isgenerated at a node, preferably through use of a voltage divider circuitwhich receives the output of the variable current. The variable voltageat the node is coupled to a control element in the unit cell, thecontrol element preferably providing a variable resistance between afirst voltage and an output node. The variable control element therebyprovides a variable current output. In this way, a first current of arelatively higher value may be utilized to control a second current of arelatively smaller value, the second current being supplied in operationto the conductive solution applied to the active electronic device forpurposes of molecular biological analysis and diagnostics. In oneembodiment, a reduction of current by a factor of 32 permits provisionof currents to the device which are easily generated and controlled, yetresults in currents of a magnitude which are required for effectiveoperation of the active biological device.

[0042] In yet another aspect of these inventions, the various devicesmay be decorated or covered with various capture sequences. Such capturesequences may be relatively short, such as where the collectionelectrode is a complexity reduction electrode. Further, relativelylonger capture sequences may be used when further specificity orselectivity is desired. These capture sequences may preferably beincluded on the collection electrodes, or intermediate transportationelectrodes.

[0043] Accordingly, it is an object of this invention to provide anactive biological device having reduced costs of manufacture yetconsistent with achieving a small size microlocation.

[0044] It is yet another object of this invention to provide deviceswhich provide increased functionality.

[0045] It is yet a further object of this invention to provide deviceswhich achieve a high degree of functionality and operability with fewerparts than known to the prior art.

[0046] It is yet a further object of this invention to provide deviceswhich are easier to manufacture relative to the prior art.

[0047] It is yet a further object of this invention to provide circuitryand systems which eliminate or reduce the pin limitation or pin outlimitations.

[0048] It is yet a further object of this invention to provide a systemwhich provides for precise current control in an active electronicdevice adapted for molecular biological analysis and diagnostics, whichmay interface with larger currents generated by a control system.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049]FIGS. 1A and 1B show an active, programmable electronic matrixdevice (APEX) in cross-section (FIG. 1A) and in perspective view (FIG.1B).

[0050]FIG. 2 is a plan view of an embodiment of the invention whichutilizes varying sized electrode regions and focusing electrodes,variously referred to as the bug chip.

[0051]FIG. 3 is a plan view of an embodiment of the invention whichutilizes a concentration electrode and paired return electrode, which isespecially useful in methods which effectively transport chargedbiological material in a wave or sweeping motion across microlocations.

[0052]FIG. 4 is a plan view of an embodiment of the invention whichutilizes a substantially circular arrangement, with a substantiallycentrally disposed concentration electrode.

[0053]FIGS. 5A, 5B and 5C show perspective views and FIG. 5D shows across-sectional view of a flip-chip system, FIG. 5A showing theunderside of the system, FIG. 5B showing a perspective view of top ofthe flip-chip structure including sample chamber, FIG. 5C showing a topperspective detail of the via, and FIG. 5D showing a cross-sectionalview of the flowcell.

[0054]FIGS. 6A and 6B show perspective and cross-sectional views,respectively, of a flip-chip system in one embodiment.

[0055]FIGS. 7A and 7B shows side and plan views, respectively, of anedge illuminated system in one embodiment of this invention.

[0056]FIG. 8 is a microphotograph of barrier wall for the Norland 83Hdam using a 1300 J/s fiber bundle source shadow masked with the flexcircuit (Flex polyimide removed).

[0057]FIG. 9 is a block diagram drawing of a multiple unit cell arraysystem.

[0058]FIG. 10A is a circuit diagram of a functionalized unit cell usablewith the system of FIG. 9.

[0059]FIG. 10B is a voltage/timing diagram for the circuit of FIGS. 9and 10A.

[0060]FIG. 10C are current diagrams as a function of time for thecircuit of FIGS. 9 and 10A.

[0061]FIG. 11 is a component level circuit diagram of a unit cell usablewith the system of FIG. 9.

[0062]FIG. 12 is a component level circuit diagram of a unit cellincluding additional test circuitry usable with the system of FIG. 9.

[0063]FIG. 13 is a schematic diagram of a circuit for providing currentcontrol in an active electronic device.

[0064]FIG. 14 is a component level circuit diagram of current mirrors.

[0065]FIG. 15 is a component level circuit diagram of column selectioncircuitry.

[0066]FIG. 16 is a component level schematic diagram for a row selectcircuit.

[0067]FIG. 17 is a plan view of a physical layout of a unit cell.

[0068]FIG. 18 is a plan view of a layout of a portion of the 20×20 testsite unit.

[0069]FIG. 19 is a block diagrammatic view of the overall control andtest system in one aspect of this invention.

[0070]FIG. 20 is a schematic block diagram view of the interconnectionbetween an input system and a probe card for connection to an activebiological matrix system.

[0071]FIG. 21 is a graph of hybridization as a function of specific andnon-specific hybridization for field-shaping and for no use of fieldshaping.

[0072]FIG. 22 is a graph of Average MFI/s at various concentrations forthe embodiment of FIG. 2, at various concentrations RCA5 BTR Reporter in5 mM histidine, showing Specific/Non-Specific Binding After Washing.

[0073]FIG. 23 is a graph of current linearity showing the electrodecurrent output in nanoamps as a function of current n in microamps.

DETAILED DESCRIPTION OF THE INVENTION

[0074]FIGS. 1A and 1B illustrate a simplified version of the activeprogrammable electronic matrix (APEX) hybridization system for use withthis invention. FIG. 1B is a perspective view, and FIG. 1A is across-sectional view taken in FIG. 1B at cut A-A′. Generally, asubstrate 10 supports a matrix or array of electronically addressablemicrolocations 12. For ease of explanation, the various microlocationshave been labeled 12A, 12B, 12C and 12D. A permeation layer 14 isdisposed above the individual electrodes 12. The permeation layerpermits transport of relatively small charged entities through it, butreduces or limits the mobility of large charged entities, such as DNA,to preferably keep the large charged entities from easily contacting theelectrodes 12 directly during the duration of the test. The permeationlayer 14 reduces the electrochemical degradation which would occur inthe DNA by direct contact with the electrodes 12, possibly due, in part,to extreme pH resulting from the electrolytic reaction. It furtherserves to minimize the strong, non-specific adsorption of DNA toelectrodes. Attachment regions 16 are disposed upon the permeation layer14 and provide for specific binding sites for target materials. Theattachment regions 16 have been labeled 16A, 16B, 16C and 16D tocorrespond with the identification of the electrodes 12A-D,respectively. The attachment regions 16 may be effectively incorporatedinto or integrated with the permeation layers (e.g., 12A), such as byincluding attachment material directly within the permeation material.

[0075] In operation, reservoir 18 comprises that space above theattachment regions 16 that contains the desired, as well as undesired,materials for detection, analysis or use. Charged entities 20, such ascharged DNA are located within the reservoir 18. In one aspect of thisinvention, the active, programmable, matrix system comprises a methodfor transporting the charged material 20 to any of the specificmicrolocations 12. When activated, a microlocation 12 generates the freefield electrophoretic transport of any charged functionalized specificbinding entity 20 towards the electrode 12. For example, if theelectrode 12A were made positive and the electrode 12D negative,electrophoretic lines of force 22 would run between the electrodes 12Aand 12D. The lines of electrophoretic force 22 cause transport ofcharged binding entities 20 that have a net negative charge toward thepositive electrode 12A. Charged materials 20 having a net positivecharge move under the electrophoretic force toward the negativelycharged electrode 12D. When the net negatively charged binding entity 20that has been functionalized contacts the attachment layer 16A as aresult of its movement under the electrophoretic force, thefunctionalized specific binding entity 20 becomes covalently attached tothe attachment layer 16A. Optionally, electrodes 24 may be disposedoutside of the array. The electrodes 24 may optionally serve as returnelectrodes, counterelectrodes, disposal (dump) electrodes or otherwise.Optionally, a flowcell may be provided adjacent the device for fluidiccontainment.

[0076]FIG. 2 is a plan view of one embodiment of the invention whichutilizes focusing electrodes 42, 44, and optionally, transportelectrodes 50, 52, 54. The device 20 includes a substrate 32, which maybe of any sufficiently rigid, substantially non-conductive material tosupport the components formed thereon. The substrate 32 may be flexcircuit (e.g., a polyimide such as DuPont Kapton, polyester, ABS orother such materials), a printed circuit board or a semiconductivematerial, preferably with an insulative overcoating. Connectors 34couple to traces 36, which in turn, couple to other electricalcomponents of the system. These components may be any form of conductor,such as copper, or gold, or any other conductor known to those skilledin the art. Various of the connectors 34 are shown unconnected to traces36 or other electrical components. It will be appreciated by thoseskilled in the art that not every connector 34, such as in a systemadapted to mate with an edge connector system will be utilized.Additionally, traces 36 may be of differing widths depending upon thedemands, especially the current demands, to be made on that trace 36.Thus, some traces 36 may be wider, such as those being coupled to thefocusing electrodes 42, 44, in comparison to those traces 36 coupled tothe microlocations within the array 38. Array 38 is preferably of theform described in connection with FIGS. 1A and 1B.

[0077] A first collection electrode 40 and counter electrodes 46 aredisposed on the substrate 32. These components generally fit within thefootprint (shown in dashed line) of the flow cell 58, and comprise arelatively large percentage thereof, preferably at least substantially40%, and more preferably substantially 50%, and most preferablysubstantially 60%. The counter electrodes 46 (sometimes functioning asreturn electrodes) and collection electrode 40 are preferably disposedat or near the periphery of the flow cell footprint 58, and maysubstantially circumscribe, e.g., to 80%, the footprint perimeter.

[0078] Typically, the collection electrode 40 and counter electrodes 46are disposed on the substrate 32 so that the electrophoretic lines offorce are significant over substantially all, e.g., 80% or more, of theflow cell volume. By way of example, the concentration 40 andcounterelectrodes 46 may be disposed near the periphery of the footprint58 of the flow cell. In yet another embodiment, they may be disposed atsubstantially opposite ends of the flowcell footprint 58 (See, e.g.,FIG. 3). In yet another embodiment, the counterelectrode substantiallycircumscribes the footprint of the flow, with a centrally disposedcollection electrode (See, e.g., FIG. 4). The relatively large percentof coverage of the flow cell footprint 58 and its position aids ineffective electrophoretic interrogation of the flow cell contents.

[0079] Returning to FIG. 2, focusing electrodes 42, 44 are disposed onthe substrate 32 to aid in focusing materials collected on thecollection electrode 40 to the array 38. The focusing electrodes 42, 44are preferably disposed in a mirror-image, “Y” or “V” shaped pattern,the open end encompassing, at least in part, the collection electrode40. As shown, there are two symmetric focusing electrodes 42, 44. Onefocusing electrode may be utilized, or more than two focusing electrodesmay be utilized. As shown, the focusing electrodes 42, 44 includesubstantially parallel portions (adjacent the array) and angled portions(adjacent the transport electrodes 50, 52, 54, and optionally, thecollection electrode 40) extending in a symmetrical manner envelopingthe transport electrodes 50, 52, 54. Stated otherwise, there are firstand second electrodes being disposed at least in part adjacent the arrayof microlocations, the distance between the first and second electrodesadjacent the array being smaller than the distance between the first andsecond electrodes in yet another region disposed away from the array.The focusing electrodes 42, 44 may optionally include portions which aredisposed on the opposite side of the array 38 from the collectionelectrode 40. The focusing electrodes 42, 44 are preferably coupled toleads 36 which are relatively larger than the leads 36 coupled to thearray 38, so as to permit the carrying of effective currents andpotentials.

[0080] Transport electrodes 50, 52, 54 are optionally included.Electrodes of monotonically decreasing size as they approach the array38 are shown. A first transport electrode 50 is relatively smaller thanthe collection electrode 40, the second transport electrode 52 isrelatively smaller than the first transport electrode 50, and the thirdtransport electrode 54 is yet smaller still. The differential sizingserves to reduce current density mismatches between locations, and aidsin reducing or eliminating burn-out which may result if too great acurrent density mismatch exists. Transport efficiently is maximized. Theratio of sizes of larger to smaller is preferably substantially 2 to 1,more preferably 3 to 1, and may be even greater, such as 4 to 1 orhigher. One field-shaping protocol is as follows: Negative Bias PositiveBias Current Bias Time Counter Electrodes 46 1st Collection 75 μA 30sec. Electrode 40 Focusing Electrodes 42, 44 1st Transport 25 μA 90 sec.(−0.2 μA) Electrode 50 1st Collection Electrode 40 Focusing Electrodes42, 44 2nd Transport 5 μA 180 sec. (−0.2 μA) Electrode 52 1st TransportElectrode 50 Focusing Electrodes 42, 44 3rd Transport 3 μA 420 sec.(−0.2 μA) Electrode 54 1st Transport Electrode 50 2nd TransportElectrode 52 Focusing Electrodes 42, 44 Row 3 1.5 μA 120 sec. (−0.2 μA)OC-80380.1 (500 nA/pad) 2nd Transport Electrode 52 3rd TransportElectrode 54 Focusing Electrodes 42, 44 Row 2 1.5 μA 120 sec. (−0.2 μA)(500 nA/pad) 2nd Transport Electrode 52 3rd Transport Electrode 54Focusing Electrodes 42, 44 Row 1 1.5 μA 120 sec. (−0.2 μA) (500 nA/pad)2nd Transport Electrode 52 3rd Transport Electrode 54

[0081] The seven steps of the field shaping protocol serve toeffectively interrogate the sample volume and to correct materials ontothe array 38 for analysis. In the first step, interrogation of thesample volume is effected through negative bias of the counterelectrodes46 and positive bias of the first collection electrode 40. The placementof the counterelectrodes 46 and collection electrode 40 generally nearthe periphery of the footprint of the flow cell 58 permit the rapid,effective interrogation of that sample volume. Secondly, with thecollected material adjacent the collection 40, that electrode is madenegative (repulsive) to materials of interest, while the first transportelectrode 50 is made positive (attractive). The repulsion and attractioneffects transport of materials from the collection electrode 40 to thefirst transport electrode 50. Additionally, the focusing electrodes 42,44 are biased negative. Such a negative (repulsive) bias serves toprovide a force which may be lateral to the direction of transport,thereby more centrally concentrating material in the solution. Thirdly,with material collected at the first transport electrode 50, thatelectrode may be biased negative (repulsive), while the second transportelectrode 52 is biased positive (attractive). The focusing electrodes42, 44 may be biased negatively, which serves to provide a repulsiveforce on the charged materials, thereby providing a transverse componentto their direction of motion and collecting the material within asmaller physical region or volume. Fourth, the second transportelectrode 52 may be biased negative, as well as optionally biasing ofthe first transport electrode 50, to effect transport away from thoseelectrodes and to the now positively biased third transport electrode54. Again, the focusing electrodes 42, 44 may retain their negativebias. The next three steps are optionally separated, as described, totransport materials to various rows or regions of the array 38.

[0082] The field shaping protocol includes currents and biased times. Inthis embodiment, there is an inversely proportional relationship betweenthe size of the electrode and the amount of current supplied to it.Further, for the collection electrode 40 and transport electrodes 50, 52and 54, there is an inversely proportional relationship between theelectrode size and the bias time, that is, the smaller the electrode,the larger the bias time. Through this protocol, the current density atvarious devices is kept relatively more uniform, optionallysubstantially similar to each other. Further, as the current from agiven electrode decreases (relative to a larger electrode) a relativelylonger bias time may be required in order to provide transport ofeffective amounts of charged material between the various electrodes.Stated otherwise, for a given amount of charged material, a relativelylonger bias time may be required to effect transport of a given amountof material at a lower current.

[0083]FIG. 3 is a plan view of another embodiment of this invention. Aswith FIG. 2, a device 60 includes a substrate 62, connectors 64, traces66 and an array of microlocations 68. The comments made for FIG. 2 andothers apply to corresponding structures in other figures. Further, thetraces 66 leading from the upper left portion of the array 68 have beentruncated for drawing simplicity. A corresponding arrangement to thoseshown in the lower right of the drawing would apply. The traces 66 maybe of the same width or of varying width, such as where a relativelywider trace 66 may be utilized for larger current carrying capacity(e.g., traces 66 to first collection electrode 70 and second collectionelectrode 72.

[0084]FIG. 3 departs from FIG. 2 in the inclusion of a first collectionelectrode 70, being disposed at least in part adjacent the array 68. Inthe embodiment of FIG. 3, first collection electrode 70 is a trapezoid,which has a long base 70 b adjacent to and parallel to one side of thearray 68 and top 70 t, which is preferably shorter than the base 70 b,with sloping sides 70 s, tapering wider (away from each other) towardthe base 70 b. The second collection electrode 72 is disposed on theother side of the array 68, and is similarly (though not necessarilyidentically) shaped and sized. Top 72 t is preferably shorter than base72 b, and accordingly, the sides 72 s are non-parallel and slope awayfrom each other, moving towards the array 68. Optionally, the electrodes70, 72 may be of different sizes, such as where the area of the firstcollection electrode 70 is approximately 10% smaller (optionallyapproximately 20% smaller) than the second collection electrode 72.Input port electrode 74 and port electrode 76 are optionally included onthe substrate 62, within the footprint of the flow cell 78. The inputport electrodes 74 and port electrode 76 are either of the same size orof different size.

[0085] In operation, the flow cell contents are interrogated by placingor biasing one of the first and second collection electrodes 70, 72attractive (typically positive) to the materials to be collected. Oncecollected, the materials may be transported away from the firstcollection electrode 70 towards the array 68. The materials may beeffectively held in place over the array 68, such as by application ofAC fields such as at a frequency in the range from 0.01 to 10⁶ Hz, mostpreferably between 0.1 to 10³ Hz between the electrodes 70, 72. Thenmaterials may be transported to the other electrode 70, 72 or may berepeatedly reacted by moving materials from the array 68 to theelectrodes 70, 72. Optionally, the microlocations of the array 68 may beelectrically active or passive.

[0086]FIG. 4 is a plan view of a concentric ring electrode embodiment.The device 80, substrate 82, connectors 84, traces 86 and array 88 areas previously described, with the exception that the array 88 may bearranged concentrically. A concentric return electrode 90 and centralconcentration electrode 92, preferably round, coact to concentratematerial at electrode 92, and then to move it over or position it abovethe array 92. As with the preceding FIGS. 2 and 3, the traces have beenshown in a truncated manner.

[0087] In the embodiments of FIGS. 2, 3 and 4, capture sequences orprobes may be disposed on the devices. Preferably these are at least onthe collection or concentration electrodes. Optionally, differentsequences are disposed on different devices such as the transportelectrode 50, 52 and 54 of FIG. 2. For example, each sequence as anapproach is made to the array may be more specific.

[0088]FIGS. 5A, 5B, 5C and 5D show views of the bottom, the top, the topwith via 109 exposed, and a side view of the system through cut A-A′ inFIG. 5B, respectively, of a flip-chip system. A device 100 includes asupport substrate 102 having a first surface 104 (optionally called thetop surface) and a second surface 106 (optionally called the bottomsurface), which may be of materials suitable for the function of supportand conduction, such as flex circuitry, printed circuit board,semiconductive material or like material. Contacts 108 lead to traces110, which lead to the second substrate 112. This second substrate 112may also be referred to as the flipped chip. This second substrate 112may optionally be a chip, system or support on which assays or otherdiagnostic materials are provided. Contacts, such as bump contacts,e.g., solder bumps, indium solder bumps, conductive polymers, or silverfilled epoxy, provide electrical contact between traces 110 and the chipor substrate 112. A sealant is disposed between the second (bottom)surface 106 of the support substrate 102 and the first (top) surface 114of the second substrate 112. Generally, the opposing faces of thesupport substrate 102 and second substrate 112 are those which areplaced in fluid-blocking contact via the inclusion of a sealant. Aninlet port 120 may be in conductive relation to a sample chamber 122,which yet further leads to the assay chamber 124, and on to the outletport 126. FIG. 5C shows a perspective view of the support 102 and thevia 128 formed through it. As shown, the lateral width of the via 128 isless than the lateral width of the second substrate 112. The secondsubstrate 112 is shown in dashed lines, which is disposed below thesubstrate 102 in the view of FIG. 5C.

[0089] In the preferred embodiment, the device 100 is formed of aminimum number of components to reduce cost, improve manufacturingsimplicity and reliability or the like. One embodiment is achieved insubstantially five components. While the device may be fabricated withfive components, the addition of components which do not detract from orvary the inventive concept may be utilized. These components are asfollows. First a support substrate 102 having a first surface 104 andsecond surface 106, and a via 128 between the first surface 104 andsecond surface 106 to permit fluid flow through the substrate 102, thesecond surface 106 supporting electrical traces. Second, a secondsubstrate 112 including at least a first surface 114, the first surfacebeing adapted to be disposed in facing arrangement with the secondsurface 106 of the first substrate, the second substrate 114 includingelectrically conductive traces connecting to an array of microlocations(See, FIGS. 1A and 1B), the array being adapted to receive said fluidthrough the via 128. Third, electrically conductive bumps 128interconnecting the electrical traces on the second surface of thesupport substrate and the electrical traces on the first surface 106 ofthe second substrate. Fourth, a sealant 130 disposed between the secondface 106 of the support substrate 102 and the first face 114 of thesecond substrate 112, said sealant 130 providing a fluidic seal by andbetween the first substrate 102 and the second substrate 112. Fifth, aflowcell is optionally disposed on the first surface 104 of the firstsubstrate 102. While the number of elements may vary, advantages may beobtained from selection of these five elements.

[0090] In operation, a sample is provided to the inlet port 120 andpassed to the sample chamber 122. The sample chamber 122 may serve tohouse various sample processing functions, including but not limited tocell separation, cell lysing, cell component separation, complexityreduction, amplification (e.g., PCR, LCR, enzymatic techniques), and/ordenaturation). Thereafter, the sample flows to the assay chamber 124.Solution containing sample flows down through via 128 (which is obscuredin FIG. 5B by assay chamber 124, though may be seen in FIG. 5C and SD).A space is formed comprising the via 128, bounded on the bottom by thesecond substrate 112, with sealant or adhesive 130 forming a barrierbetween the interface of the second surface 106 of the support substrate102 and the first surface 114 of second substrate 112.

[0091] In the preferred method of manufacture, a light curable sealantis wicked or otherwise provided to the interface between the secondsurface 106 of the support substrate 102 and the first surface 114 ofthe second substrate. Light is provided through the via 128. A dam isformed, stopping the advance of the sealant, thereby maintaining thearray, e.g., 18, substantially free from sealant or adhesive. (See FIG.8 for a microphotograph showing the sealant free area of the array, thecured leading edge of the darn and sealant on the exterior portions ofthe device.) By the appropriate sizing of the lateral width of the via128, the via 128 serves essentially as a shadow mask fr the incidentlight, which serves to cure the sealant. Alternatively, the sealant maybe supplied to the interface between the second surface 106 of thesupport substrate 102 and the first surface 114 of the second substrate112 in an amount and with a viscosity such that it does not flow ontothe array 18. Further or final curing of the sealant may be performed asrequired, such as by heating.

[0092]FIG. 6A shows a perspective exploded drawing and FIG. 6B shows across-sectional view, respectively, of a flip-chip system in accordancewith one implementation of this invention. The system of FIGS. 6A and 6Binclude an edge illumination member 140, unique sample chamber 134design, and a ‘butterfly’ input and output chamber design as compared toFIGS. 5A, 5B and 5C. A chip or substrate 130 has a first surface 130 tand a second surface 130 b, at least the first surface 130 t includingelectrical regions or traces 132 thereon or therein. While theembodiment shown in cross-section in FIG. 6B shows the trace 132disposed on the top surface 132 of the chip or substrate 130, theelectrical regions may be contained wholly or partially within the chipor substrate 130, such as through the provision of semiconductiveregions. These semiconductive regions may be controlled in an activemanner so as to provide selective connections within the chip orsubstrate 130. Typically, the first surface 130 t is that surface onwhich the active biological interactions will take place. Optionally, anedge illumination member 140 may be disposed adjacent to andsubstantially coplanar with the first surface 130 t of the chip orsubstrate 130. The illumination sheet 140 preferably includes holes,vias or pathways 144 to permit electrical interconnections 156 to passtherethrough. As can be seen, the illumination sheet 140 may be disposeddirectly over conductive traces 132 or may be directly affixed to theadjacent supporting sheet 150. Electrical traces 132 may be included onthe first surface 130 t of the substrate or chip 130. An electricallyconductive element 136, such as a solder connection, indium bump,conductive polymer or the like couples the conductive pathway 132 on thesubstrate 130 to the conductive portion of the contact trace 154. Thecontact trace preferably is then contacted by a conductive member 156,such as a wire, whisker wire, or other electrical contact, forconnection to the remainder of the circuit. Sealant 180 is preferablydisposed between the substrate or chip 130 and the next layer 150, suchas the flex support layer.

[0093] In FIG. 6B, the drawing has been presented with a conductivemember 136 on the left hand side, but with sealant 180 on the right handside. It will be appreciated that other conductive members 136, notdisposed in the plane of the cut, are included and provide furthermechanical support between the substrate 130 and the trace support layer150. Further, the edge illumination layer 140 includes a terminal edge142, which is disposed toward the upper surface 130 t of the substrateor chip 130. The edge illumination layer 140 may terminate outside of orinside of the sealant 180. An adhesive layer 160 is disposed adjacentthe trace support layer 150, and provides adhesive contact to an upperlayer 170. The upper layer 170 may optionally include pathways,indentations, or other cutouts, such as shown for an inlet 176 and anoutlet 176′. As shown, the adhesive layer 160 may optionally be adie-cutable adhesive material, such as one which includes release paperon both the top surface and the bottom surface prior to assembly.Suppliers of such materials include 3M or Dupont. As shown in FIG. 6A,the die-cutable adhesive material 160 may be cut so as to form all or apart of the wall 162, 164 of a chamber. As shown, the geometry may bemade in any desired shape or flow cell configuration.

[0094] Preferably, a top member 170 is provided. As shown, the topmember 170 may extend substantially over the remainder of the device.Optionally, the top member 170 may form a window 172 or othercontainment surface at the top of the flow cell chamber. Preferably, thetop material is formed from polycarbonate or polystyrene. In theconfiguration of FIGS. 6A and 6B, it is typically contemplated that thearray of test sites will be accessed optically through the top member172, and accordingly, it is desirable to form the top member frommaterials which are substantially transparent to both the excitation andemission radiation.

[0095]FIGS. 6A and 6B show one geometry for a flow cell. In this‘butterfly’ configuration, the inlet 176 is connected to a firstexpanding region 174 wherein the sidewalls of the chamber start at afirst dimension d and expand, preferably monotonically, and mostpreferably linearly, to a dimension D at a point closer to the flow cellchamber 134. The flow cell chamber 134 region is characterized bysubstantially parallel sidewalls 166. Preferably, a first decreasingwidth region is provided between the flow cell region and the output.Most preferably, the decreasing region begins with a width D′, mostpreferably where D′=D, and decreases to a width d′, preferably whered′=d. As can be seen in FIG. 6A and 6B, the height of the inlet chamber174 decreases from the inlet at a height H to a lesser height h at theinlet to the flow cell chamber. Preferably, the decrease is monotonic,and most preferably, linear.

[0096] In the preferred embodiment, the height h of the inlet chamberand the width w are chosen such that a substantially constant flow areais provided, that is, the product of the height h and the width w (h×w)is substantially constant. Thus, as shown in the combined view of FIG.6A and 6B, at the portion of the inlet chamber adjacent the inlet, whilethe height h is relative large, the width w is relatively small.Correspondingly, when proceeding through the inlet chamber towards theflow cell chamber, as the width w increases, the height h decreases.Preferably, the outlet chamber includes substantially the same geometry,and preferably the same flow cell area constant.

[0097]FIGS. 7A and 7B are cross-sectional and plan views, respectively,of an edge illuminated, flip-chip system in accordance with oneembodiment of the invention. To the extent possible, a consistentnumbering of elements from FIGS. 6A and 6B will be utilized. A supportsubstrate 150 is generally planar, and includes a first face 150 t and asecond face 150 b. A via 126 (shown in dashed lines for thecross-section) permits fluid or solution flow from above the supportsubstrate 150 to the second substrate 130, particularly to the firstsurface 130 t of the second substrate 130. Sealant 180 is providedbetween the second face 150 b of the support substrate 150 and thesecond substrate 130. The sealant 180 provides a preferably fluidtight-seal, so as to permit fluid flow to the array on the secondsubstrate 130. A source of illumination 190, such as a laser bar,illuminate the array on the second substrate 130. Preferably, the systemincludes a waveguide 140 with an input 146 adapted to receiveillumination from the source 190, and to provide illumination via output142. The waveguide 140 is preferably co-planar with the supportsubstrate 150, and may be secured to it, such as by being adhered to thesecond surface 150 b of the support substrate 150. Electronics 192 maybe included to control the system. Optionally, surface mountedelectronic components may be included on the substrates 130, 150.Fluidics 194 may be provided in combination with the system to aid inprovision of the sample to the second substrate 130.

[0098]FIG. 9 is a block diagrammatic depiction of a multiple unit cellarray. In the preferred embodiment, a system or chip includes amulti-site array 210 with electrically repetitive site cell locations.Typically, the array is formed of rows and columns, more typically anequal number of rows and columns, yet most typically in an orthogonalarrangement for rows and columns. For example, an array of 10×10, 20×20or more may be formed with these techniques. The individual unit cell212 of the array 210 of unit cells is selected by action of selectorssuch as a row selector 220 and a column selector 230. The selectors 220,230 may be a memory, such as a shift register memory, or a decoder, or acombination of both. An input for address information receivesaddresses, typically from off-chip, though on chip address generatorsmay be utilized. In the preferred embodiment, the row selectors 220comprise shift registers, either in a by one configuration (x1), or in awider configuration, such as a by four configuration (x4). In operation,the selection registers are sequentially loaded with values indicatingselection, or not, of a unit cell 212, and optionally, the value ofoutput for that cell. Optionally, memory may be provided to retain thosevalues so as to continue the output from the unit cell.

[0099] Considering FIG. 9 in more detail, an array 210 includes aplurality of unit cells 212. In the preferred embodiment, the unit cells212 are arranged in rows and columns, the designation row in FIG. 9depicting a horizontal arrangement relative to the text, and a columndesignating a vertical arrangement relative to the text (though it willbe understood by those skilled in the art that the designations row andcolumn may be reversed). The designation row or column may also refer toa group or subset of unit cells 212, such as a portion of a row orcolumn, or a group or set of unit cells 212 which are not linearlycontiguous. In general, there are m rows and n columns of unit cells212, typically where m=n, and m=2, 3, 4 . . . . By way of example, a 5×5matrix of unit cells 212, a 10×10 matrix of unit cells 212 and a 20×20matrix of unit cells 212 provides for a total number of unit cells of25, 100 and 400, respectively.

[0100] In FIG. 9, various levels of complexity of unit cell 212 areshown. The uppermost depicted unit cell 212 is depicted as a singleblock-diagram unit, whereas the unit cell 212 disposed central to thefigure is shown in greater complexity, akin to the structure disclosedand described in more detail in FIG. 10. It will be understood thatthese alternatives are depicted for expository convenience and variety,and that in a typical implementation, the construction of the individualunit cells 212 will be the same for a given device.

[0101] The unit cells 212 are addressed by action of at least one rowselector 220 and at least one column selector 230. This detaileddescription begins with the case of a single row selector 220 and columnselector 230, and later describes the use of additional selectors 220′,230′. Row selector 220 receives input information 222 and outputs a rowselection signal 294 (see FIG. 10A) on one or more row lines 224. Theselection signal on the row line 224 is supplied to the unit cell 212,and interacts therewith such as through a row contact 226. As drawn, aportion of row line 224 is shown disposed to the left and a portionshown drawn to the right of the unit cell 212 centrally disposed in thearray 210. In typical implementation, the row line 224 will beelectrically continuous, though may be made of any combination ofmaterials. For example, the row line may be one continuous conductiveline, such as formed of conductive polysilicon, or may be a combinationstructure such as where conductive segments are electrically connectedvia a higher conductivity material, such as metal, such as aluminum.

[0102] The column selector 230 receives an input 232 for determining theselection of a column, or in the preferred embodiment, the value (orcorrelated value) of the output at the unit cell 212. The columnselector 230 is coupled to the column lines 234 which serves to providea column select signal 296 a-d to the unit cells 212. In the preferredembodiment, the column selector 230 selects more than two states (e.g.,four states 296 a-296 d), preferably voltage states, which are suppliedvia the column line 234 to the unit cell 212. The column line 234 iscoupled to the unit cell 212, such as through a column contact 236. Inthe preferred embodiment, the column contact may be a control gate for atransistor, such as a field effect transistor. (See, e.g., FIGS. 11 and12).

[0103] If required for activation of the unit cell 212, a second rowselector 220′, input lines 222′, second row lines 224′ and columncontacts 226′ may be included. Likewise, a second column selector 230′may be added, having an input 232′, and being coupled to secondary orsupplemental column lines 234′ which in turn are coupled to secondarycolumn contacts 236′.

[0104] As shown, the row selectors 220, 220′ and column selectors 230,230′ optionally include an enable input 228, 228′ or chip select 238,238′. One of the functions of these signals is to permit entry of inputinformation 222, 222′, 232, 232′ without the activation of a row line224, 224′, or column line 234, 234′. Further, some or all of the rowselectors 220, 220′ and column selectors 220, 220′ and column selectors230, 230′ may include an output 229, 229′, 239, 239′ which may be usedfor output of information. In one application, the output value may be asignal or bit, such as the most significant bit of a series, indicatingthat the input data has been successfully loaded into the row selector,220, 220′ or column selector 230, 230′. Optionally, this outputinformation may be utilized to then trigger the enable or chip selectsignals 228, 228′, 238, 238′.

[0105] Within the level of detail of FIG. 9, the row selectors 220, 220′and column selectors 230, 230′ function to receive row and column inputinformation 222, 222′, 232, 232′ and to use that to select one or moreunit cells 212, and optionally, to provide signal values indicative ofthe level of current (potential) to be provided from the unit cell 212.The selectors 220, 220′, 230, 230′ may be in the form of memory, such asin the form of a shift register memory (See FIGS. 15 and 16 for detail),or may be in the form of a decoder circuit, such as where the desiredaddresses are provided as input information and the output is then in adecoded relationship thereto. Numerous circuits are known to thoseskilled in the art to effect this functionality.

[0106] A current source 240, such as a current mirror, optionallyreceives a source of current 242 and a control signal 244 (VCASP).Connections 246, 246′ couple the current from the source 240 to columnselector 230, and if present, second column selector 230′. As shown, thecoupling lines 246, 246′ are separate wires (designated “a” to designatea number of wires equal to a). Further, one or more sources of current242 may be supplied. As will be described in connection with FIG. 10C,below, the value of the current may be static, or may vary over time(such as in the application of a pulsed waveform, sinusoidal waveform,square wave, sawtooth, etc.). Generally, any desired varying waveformmay be utilized.

[0107] Utilizing the structure shown in FIG. 9, each of the unit cells212 may be activated at a given time. Alternatively, certain unit cells212 may be activated and yet other unit cells remain inactive. By way ofexample, if a given column has been selected at a first value, each ofthe unit cells within that column which are associated with one or moreselected rows selected by the row selector 220 will be activated at thevalue corresponding to that level of the voltage on the column. Yetother unit cells within that same column may be placed at the same or adifferent level by coupling to the second column selector 230′, wherethe one or more row lines associated with those unit cells are driven bythe second row selector 220′. Thus, within one column of unit cells,each unit cell 212 may be either driven at a value corresponding to thesignal on the column 234 associated with the column selector 230, orwith the value on the column associated with the second column selector230′, or be in an undriven, unconnected, floating or high impedancestate. In a like manner, other columns may be set to desired levels ofoutput. In this way, the entire array of unit cells may be placed in thedesired state or set of states. The use of the terms ‘levels of output’and ‘desired state’ include signals which vary as a function of time.Additionally, more values within a given column 234, 234′ may be added,such as through the addition of further column selectors and columnlines which are coupled to selected unit cells 212.

[0108]FIG. 10A shows a schematic block diagram of a unit cell 212 andreturn electrode 250. To the extent possible, the numbering conventionin FIG. 10 corresponds to that adopted in FIG. 9. A variable currentcontrol element 260 includes an input 262, an output 264 and a controlelement 266. The control element 266 is coupled to a line, such as thecolumn line 234, which is in turn coupled to the column select 230. Aselector switch 270 includes an input 272, an output 274 and a controlelement 276. The control element 276 is coupled to a control line, suchas a row line 224. The output 264 of the variable current controlelement 260 is coupled to the input 272 of the select switch 270. Theoutput 274 of the select switch 270 couples to a node 280 which providesthe output current 282, I_(out). A first potential 284, e.g., Vcc, isprovided to input 262 of the variable current control element 260.

[0109] In operation, application of a signal on row line 224 to theinput 276 of the select switch 270 provides a conductive path betweennode 280 and output 264 of the variable current control element 260. Thesignal value applied to the column line 234, which is coupled to theinput 266 of the variable current control element 260 serves to providea variable amount of current flowing through the series connectedvariable current control element 260 and select switch 270 between thefirst potential node 284 and the output node 280. A return electrode 250serves to complete the circuit, though it will be appreciated that thereturn electrode 250 may be yet another unit cell 212.

[0110] In the preferred embodiment, the variable current control element260 is a transistor, such as a field effect transistor, and mostparticularly a MOSFET. The select switch 270 is preferably a transistor,more preferably a field effect transistor, and most particularly aMOSFET. Various types of particular implementation may be utilized,whether C-MOS, N-MOS, CMOS, bipolar, gallium arsenide, or otherwise, solong as consistent with the functional requirements of the system.Further, in the preferred embodiment, a matching arrangement of a secondvariable current control element 260′ and second select switch 270′couples between a second potential 284′ and the output node 280.Optionally, channel lengths of the various devices may be arranged suchthat a symmetric arrangement is implemented. For example, in a CMOSimplementation, the p-channel select device may have a shorter channellength than the n-channel device, to compensate for the differingelectron/hole mobility. (e.g., 80μ v. 126μ channel length). A similarnumbering scheme has been adopted with the addition of primes. Thediscussion regarding the circuit, above, applies to the circuitryincluding the second variable current control element 260′ and secondselect switch 270′.

[0111]FIG. 10B shows signals as a function of time for exemplary controlsignal of the unit cell 212. The generation of the output signal 290indicates the completion of the entry of the data into the selectors220, 220′, 230, 230′. The output signal 290 may then be used to triggeror activate the enable signal 292. The enable signal 292 in turn maypermit the select signals for the row select 294 and column selectsignals 296A, 296B, 296C and 296D pass to the unit cell 212. Asdepicted, a single row select signal 294 is provided, wherein thatsignal is provided to a select circuit 270, 270′ having preferablybistate operation. The column select signals 296A, 296B, 296C and 296Dmay be of differing values, preferably of more than two values, in thepreferred embodiment comprising at least four values, which are thenprovided to the inputs 266, 266′ of the variable current controlelements 260, 260′. As explained further, below, these values may bestatic or dynamic.

[0112]FIG. 10C depicts exemplary values of current (or voltage) as afunction of time which may be supplied to the electrodes. In oneembodiment, a static, direct current (sourced or sinked), which does notvary as a function of time may be supplied. While the value of thecurrent is static, it will be understood that the selection, typicallydigital selection, of whether to permit this current to drive theelectrode or not is utilized, such that the electrode is selectivelydriven as a function of time. The second waveform in FIG. 10C shows asquare wave. The square wave may be for unit directional current orbi-directional current. An offset bias may be utilized as desired. Asshown in the third waveform in FIG. 10C, the waveform may have aperiodicity which has a subcomponent waveform included within it. Thefourth waveform in FIG. 10C shows a generally sinusoidal waveform. Thefifth waveform in FIG. 10C shows a sawtooth waveform. It will beappreciated that any waveform consistent with the goals and objects ofthis invention may be utilized in conjunction with the devices andmethods disclosed herein. By supplying a waveform, most particularly, acurrent waveform, which is selectively controllable by digital selection(such as through the action of the row selector 220 in FIG. 9), a highdegree of flexibility and control is achievable. Further, the waveformssupplied to various test sites within the device need not be the same.For example, the first column may have a static, direct current waveformapplied to it, the second column may have a square wave waveform appliedto it, whereas the third column has a sinusoidal waveform applied to themicrolocations in that column as selected by the row selector, and soon.

[0113] The waveforms for the current may be generated on the chip or offchip. In practical implementation, the waveforms may be generatedthrough the use of digital to analog converters (DACs), digital signalprocessors (DSPs), variable current waveform generators, on-chipmemories, all optionally under control of a control system utilizing acentral processing unit (CPU) or other version of microprocessorcontrol.

[0114]FIGS. 11 and 12 are circuit schematics for a driving circuit for aunit cell in one embodiment of this invention. FIG. 12 expresslyincludes test circuitry, such as test transistors 320, 330, whereas FIG.11 does not. The common aspects of the figures will be describedtogether.

[0115] In one preferred embodiment of a unit cell 212, a symmetricarrangement is utilized. A first column select unit 260, preferably atransistor, and a first row select unit 270, also preferably atransistor, are in series relation between a first source 284, e.g.,voltage and/or current source, and a node 280, typically a currentoutput node. In the preferred embodiment, the column select transistor300 may be precisely controlled under application of a gate voltage suchas from the column shift register memory (See FIG. 15). Preferably, theselect units 260, 260′ may differ from each other in theircontrollability, such as by varying the channel length in the controltransistor. Thus, by application of potentials from the row selector220, 220′ and column selector 230, 230′, application of potential to thecontrol gates 302, 312 results in output of current 282 at the unitcell.

[0116] The unit cell circuit 212 may further include a second columnselect unit 270′, preferably a transistor 300′, and a second row selectunit 270′, also preferably a transistor 310′, used in series relationbetween a second source 284′, e.g., voltage and/or current source, and anode, typically the previously referred to node 280, i.e., a currentoutput node. In the preferred embodiment, the first source 284 is asupply potential Vcc and the second source 284′ is a referencepotential, such as ground. Preferably the nodes are the same node 280,such that there is a series connection between Vcc 284 and ground 284′of the first column select unit 260, 260′ and first row select unit 270,the node 280, and the second row select unit 270′ and the second columnselect unit 260′.

[0117] In yet another form of operation of the circuit, oralternatively, a different mode of operation of the circuit shown inFIG. 11, the circuit may be tested for continuity by simultaneouslyactivating each of the first and second row and column selecttransistors 260, 270, 260′ and 270. In this way the source 284 and sink284′ are directly conductively connected.

[0118] In yet another aspect of the preferred embodiment, test circuitryis included. FIG. 12 shows a schematic diagram of such a system. A firsttest transistor 320 spans the first column select transistor 260 andfirst row select 270 transistor. Likewise, a second test transistor 330spans the second column select transistor 260′ and second row selecttransistor 270′. Selective activation ensures continuity of the circuit.

[0119] While the circuitry described herein may be implemented in anyknown technology consistent with the achievement of the desiredfunctionality of this system, one preferred mode of implementation isthrough CMOS circuitry. In one implementation of the circuits of FIGS.11 and 12, the column select devices 260, 260′ include a relatively longchannel length. These relatively large field effect transistors serve toprovide more accurate current control. By way of example, oneimplementation of this circuitry is in transistors having a 6 micronchannel width. The upper column select unit 260 (controlled byVI_P_CSEL) has a channel length of 80 microns, and the lower columnselect unit 260′ (controlled by signal VIN_N_CSEL) has a channel lengthof 126 microns. The difference in channel lengths reflects thedifference in mobility of electrons and holes, and seeks to balancethese two devices. By way of comparison, the remaining devices in FIGS.11 and 12 have a 6 micron channel width, and a 4 micron channel length.Alternative implementations of the unit cell 212 include seriesconnection of transistors, including dedicated series selectiontransistors for a row select and column select, plus an additionaltransistor for output level (current or voltage) select. More broadly,any circuit which receives cite selection information (e.g., row andcolumn select) and value and/or polarity information and causes theoutputting of the desired current or potential may be utilized.

[0120]FIG. 13 shows a schematic view of a current control system usefulin the inventions disclosed herein. To the extent possible, thenumbering convention is consistent with those of other drawings. Thecircuit serves to receive an input current 340 which is selectivelycontrollable so as to generate a voltage at node 342 which is in turncoupled to a line 234 (shown to be the column line 234) which in turn iscoupled to the control element 266 of the variable current controlelement 260. The variable current control element 260, row select switch270, first supply voltage 284 and output current from node 280 are asdescribed previously. Similarly, a current controlled circuit may beutilized to control a symmetric circuit (e.g., elements 260′ and 270′ ofFIG. 10).

[0121] The input currents 340 are provided to control elements 344. Eachcurrent of a given subscript is provided to a control element 344 oflike subscript. The control element 344 serves to selectively providecurrent at the output 346. As shown, the current outputs 346 are summedsuch that the current at node 348 may be varied based upon the states ofthe switches or control elements 344 a-d. A voltage divider arrangementis then provided wherein a potential 350 is provided to resistor 352which connects to node 342. By supplying the current at node 348 to node342 and then through resistor 352, a variable voltage is provided atnode 342. Optionally, the resistor 342 may be a device which, such as atransistor, which is conductive only in the event that current will besupplied to node 348. Thus, in the event that each switch 344 a-d is toremain off, such a circuit would also not include the resistor 352 inany state of conduction in that event. (See FIG. 15 for a detailedimplementation).

[0122]FIG. 14 shows a detailed circuit diagram for a portion of thecurrent mirror for use in the system. Four identical circuits are shownin FIG. 14, and the description with respect to one circuit will applyto all circuits equally. A current node 400 couples to the output of afirst transistor 402 and second series connected transistor 404 which isin turn connected to a first potential 406 (Vdd). Optionally, thetransistors 402, 404 are biased to or connected to the supply voltage406. The control gate 408 for the second series connected transistor 404is connected to the current node 400. The current node 400 is alsoconnected to the output (source or drain) of first transistor 402. Thecontrol gate 410 of the first control transistor 402 is controlled by asignal 412. The signal 412 serves as a select signal for the currentmirror. The select signal 412 is supplied to the current mirrors tocause selective provision of the current from current nodes 400 to thecolumn select circuitry.

[0123]FIG. 15 is a detailed circuit diagram of a column select circuit(See, e.g., column selector 230 in FIG. 9). A shift register arrangementis provided by a series of flip-flops 420, the first of which receivesas an input the input information (Q(0)), optionally inverted byinverter 422. As explained in connection with FIG. 9, an optional output229 may be provided from the selector, e.g., shift register 230. Asshown, two stages, each comprising four bits, is shown for a shiftregister. In implementation, a 20×20 matrix or array of unit cells wouldrequire 80 bits in the shift register 126 if 4 bits are assigned to eachcolumn. The outputs 430 are provided as control signals to currentcontrol circuitry 432. As shown, the current control circuitry consistsof a parallel arrangement of a first transistor 434 and secondtransistor 436, of opposite conductivity type, having their controlgates coupled to the signal 430 as supplied directly to the firsttransistor 434 and through an inverter 438 to the second transistor 436.In operation, the current supplied to node 440 is then selectivelypassed to output node 442 under control of the signal 430. The currentat the output node 442 is summed with the output currents from the threeother control circuits 434 for the column position, the summingoccurring by or before node 496.

[0124] The summed current at node 496 is passed to node 492 which mayserve as a voltage tap for column line 434. Logic 440, here shown to bea NAND gate, receives as inputs the outputs of the inverters 438. Theoutputs of the inverters 438 are provided to the NAND gate 440, whichlogically serves as an OR for the various inputs. Thus, the selection ofany of the various current sources serves to activate the gatedtransistor controlled by the logic element 440.

[0125] The shift register 126 includes multiple series connectedflip-flops 420. The value signal is provided as input to the inverter422 and then to the D input of the flip-flops 420. A clock signal (CM)and chip select signal (CS) are provided. The output of the lastflip-flop 420 (right most in FIG. 15) is provided to an inverter whichprovides the output bit at node 424.

[0126]FIG. 16 shows a component level schematic for a shift register450. Flip-flops 452 receive an input 454 (Q(0)) which is passed to otherflip-flops 452 via the Q output of one flip-flop to the D input of thenext flip-flop 452. Optionally, an output 456 provides an indication ofthe most significant bit (or other indicator of loading) from the shiftregister. The enable signal 460 is provided as input to logic 462 (shownhere to be a NAND gate) which also receives as input the output (Q pin)of the associated flip-flop 452. The output of logic 462 controls passcircuitry 464 which serves to selectively pass the signal 466 which ifpassed through circuitry 464 constitutes the row select signal 468. Thecircuitry repeats for the number of stages in the shift register 450,and the description provided here applies to those stages.

[0127]FIG. 17 shows the layout for one implementation of a unit cell.Column lines 234, 234′ are shown running vertically, which couple to thecolumn selectors (See FIG. 9). Row lines 224, 224′ are shown runninghorizontally. The supply voltage VDD 500 and the second voltage 202 (VSSe.g., ground) are disposed running generally parallel to the columnlines, 234, 234′. The optional test control lines 504, 504′ providecontrol signals for the n test and p test circuitry, respectively. Rowline 224 is connected by conductive member 506 to gate 508 whichoverlies the channel region underneath. Likewise, the row line 224′couples to gate 508′ which overlies the channel region for the selecttransistor. The column lines 234, 234′ are electrically coupled to gates510, 510′ which overlie the channel region which are then coupled to thefirst supply voltage VDD 500 and second supply voltage VSS 502,respectively. The channel length underlying the gates 510, 510′ differ,the difference in length being selected such that the operative deviceshave similar suitable properties. The output of the switchingtransistors controlled by gates 508, 508′ are provided throughconductive member 512 to electrode 514.

[0128]FIG. 18 shows a plan view of a portion of a 20×20 array of unitcells. FIG. 18 shows a portion of the overall chip, recognizing that thestructures such as the unit cells, shift registers, row and columndecoders and current mirrors are typically repeated identicallythroughout the chip. A plurality of unit cells (shown in detail in FIG.17) are included. Counter or return electrodes 520 are preferablydisposed at the periphery of the array of the unit cells. The electrodes520 preferably encompass or circumscribe the unit cell array.Optionally, multiple electrodes may be utilized to encompass the array.In the preferred embodiment, 4 L-shaped electrodes bracket the array (acorner of one being shown), each electrode bracketing substantially ¼ ofthe array. These electrodes may be utilized to move undesired materials,and to serve as a dump or disposition electrode. Row selectors 220″ (solabeled to correspond to the numbering in FIG. 9) are disposed to theright of the array and electrode 520 in FIG. 18). Column selectors 230″are disposed exterior to the array and the electrode 520. Current mirrorcircuitry 240″ is optionally disposed at the comers of the chip. Theselection and arrangement of components on chip is made to optimize thefunctionality of the device. Inclusion of components on chip, which istypically the disposable component, permits local control offunctionality, though with increased device costs. While variousarrangements are possible, the structure shown in FIG. 18 is thepreferred embodiment for the 20×20 chip.

[0129]FIG. 19 is a schematic diagram of the overall system. A controlcomputer 530 is coupled to a test board 532 and probe card 534 via buses540. Optionally, a connector, such as an RS232 connector is utilized.The probe card 534 interfaces with the actual active electronic device.The output from the device may be provided to receiving systems 542,which may include analog to digital converters for provision of digitaldata via the bus 540 to the computer system 530.

[0130]FIG. 20 is a expanded block diagram of the test board and probecard of FIG. 19. The serial port connection 550 couples to a universalasynchronous receiver transmitter (UART) 552 onto a controller 554. Businterconnection then couples to current sources 556 and current sinks558. Various digital to analog converters such as the dump DACs(digital-to-analog converters) 560 and bias DAC 562 are provided. Shiftregisters 264 coupled to the probe card 534. Analog to digitalconverters 556 may receive an output signal, such as from the probe card534. If the shift registers include an output (See output 229, 229′,239, 239′ and FIG. 9) a shift register loopback 568 may be provided.

[0131]FIG. 21 shows a graph of electronic hybridization utilizing thechip of FIG. 2. The graph shows the fluorescent intensity, in MFI/s as afunction of column number. The three bar graphs labeled column 1, column2 and column 3 utilize field shaping, and show specific hybridization onthe left bar graph in comparison to non-specific hybridization on theadjacent right hand column. The three couplets of bar graphs labeledcolumn 1, column 2 and column 3 above the designator “standard” show thesame system but without field shaping. The discrimination betweenspecific versus non-specific binding is significantly less than in thecase where field shaping is utilized. The sequences wereATA5/ATA7/biotin, and 10 pM RCA5/BTR.

[0132]FIG. 22 shows a graph of experiments performed with the system asshown in FIG. 2. The y-axis shows the average MFI/second, and the x-axisshows various rows of various concentrations. The first couplet ofparagraphs shows a 50 niM concentration of RCA5 BTR reporter in 50 mMhistidine, and depicting the specific/non-specific binding afterwashing. The first couplet shows rows 1 and 2 comparing the specificbinding (ATA5/RCA5) to the non-specific binding (ATA7/RCA5), showing a12:1 and 50:1 improvement. The middle couplets of bar graphs show a 50pM concentration of RCA5 BTR reporter and shows a 3.9:1 and 4.9:1 ratioof specific binding to non-specific binding signal intensity. The lastset of couplet bar graphs shows a 1 pM concentration of RCA5 BTRreporter and shows a 4.4:1 and 4.0:1 ratio of specific binding tonon-specific binding.

[0133]FIG. 23 is a graph of current linearity showing the electrodecurrent output in nanoamps as a function of current input in microamps.A legend is provided to indicate the various lines on the graph.

[0134] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

We claim:
 1. A circuit for control of an output current in an activebiological control reaction system, comprising: a first column selecttransistor, the first column select transistor being adapted for controlby a column selector, a first row select transistor, the first rowselect transistor being adapted for control by a row selector, the firstselect transistors being connected in series to each other and between anode and a first supply, an output connected to the node, a secondcolumn select transistor, the second column select transistor beingadapted for control by a column selector, and a second row selecttransistor, the second row select transistor being adapted for controlby a row selector, the second select transistors being connected inseries to each other and between the node and a second supply.
 2. Thecircuit of claim 1 for control of an output current in an activebiological control reaction system wherein the output is directlyconnected to the node.
 3. The circuit of claim 1 for control of anoutput current in an active biological control reaction system whereinthe row select transistors and the column select transistors are fieldeffect transistors.
 4. The circuit of claim 1 for control of an outputcurrent in an active biological control reaction system wherein thefirst and second row select transistors are CMOS transistors.
 5. Thecircuit of claim 1 for control of an output current in an activebiological control reaction system wherein the first and second columnselect transistors are CMOS transistors.
 6. The circuit of claim 5 forcontrol of an output current in an active biological control reactionsystem wherein the channel length of the column select transistors islarger than the channel length of the row select transistors.
 7. Thecircuit of claim 1 for control of an output current in an activebiological control reaction system further including a first testtransistor spanning the first supply and the node.
 8. The circuit ofclaim 7 for control of an output current in an active biological controlreaction system wherein the first test transistor is adapted for controlby a test signal.
 9. The circuit of claim 8 for control of an outputcurrent in an active biological control reaction system furtherincluding a second test transistor spanning the second supply and thenode.
 10. The circuit of claim 9 for control of an output current in anactive biological control reaction system wherein the second testtransistor is adapted for control by a test signal.
 11. The circuit ofclaim 1 for control of an output current in an active biological controlreaction system wherein the fi rst supply is Vcc.
 12. The circuit ofclaim 1 for control of an output current in an active biological controlreaction system wherein the second supply is ground.
 13. The circuit ofclaim 1 for control of an output current in an active biological controlreaction system wherein the first and second column select transistorsare controlled under application of a gate voltage from a column shiftregister memory.
 14. The circuit of claim 1 for control of an outputcurrent in an active biological control reaction system wherein thefirst and second row select transistors are controlled under applicationof a gate voltage from a row shift register memory.
 15. A circuit forcontrol of an output current in a multiple unit cell array, comprising:an array of unit cells arranged in rows and columns, wherein each unitcell comprises: a column select transistor, the column select transistorbeing adapted for control by a column selector; a row select transistor,the row select transistor being adapted for control by a row selector,the column select transistor and the row select transistor beingconnected in series to each other and between an output node and a firstsupply; and a return electrode.
 16. The circuit of claim 15 , whereineach unit cell further comprises: a second column select transistor, thesecond column select transistor being adapted for control by a columnselector; a second row select transistor, the second row selecttransistor being adapted for control by a row selector, the secondcolumn select transistor and the second row select transistor beingconnected in series to each other and between the output node and asecond supply.
 17. The circuit of claim 15 wherein the return electrodeis another unit cell.
 18. The circuit of claim 16 wherein the firstsupply is Vcc.
 19. The circuit of claim 16 wherein the second supply isground.
 20. The circuit of claim 16 wherein the row select transistorsand the ors. column select transistors are field effect transistors.